Hepatitis E virus, known previously to cause much acute viral hepatitis in developing countries, has recently been identified as a cause of sporadic hepatitis in industrialized countries. It also has been found to cause chronic hepatitis in severely immune-compromised persons such as those receiving organ transplants. Inability to propagate the virus efficiently in cell culture or in small animal models has inhibited attempts to characterize its molecular biology or identify its pathogenic determinants. Two genotypes of the virus infect humans only, but the remaining two genotypes that infect humans are zoonotic and swine are a known reservoir. It seems certain that other animal reservoirs for human HEV must exist but they have yet to be identified and the biological factors that permit some strains to cross species boundaries are completely unknown. In FY2010, we isolated a new strain of genotype 3 HEV from the stool of a chronically infected patient and serially passaged this new virus strain in cultured human hepatoma cells and adapted it to grow relatively efficiently in these cells. During the adaptation process we discovered that a recombinant HEV with a 174 nucleotide insertion as well as numerous point mutations was selected.We made an infectious cDNA clone of this virus. In FY2013, in collaboration with Andrew Firth, we mutated the infectious cDNA encoding our cell culture-adapted virus ( P6, Kernow strain, genotype 3) and showed that a highly conserved sequence served as a cis-reactive control element that required an intact stem-loop structure. In FY2012, we identified a number of non-hepatic cell lines that supported replication of P6 genomes and characterized them for their ability to be infected, produce infectious virus, and permit virus egress. This was done to identify possible substrates for producing a vaccine strain of HEV. Cross-genotype chimeric viruses were constructed which were tested for their host-range preference on human and swine cells in order to determine the basis for the zoonotic aspect of genotype 3 infections.In FY2013 we showed that genotype 1 virus infection of swine cells was inhibited at the entry step and also that translation of the genotype 1 subgenomic RNA was abnormal in these cells. In FY2013 we also showed that addition of the 174 nucleotide insertion from genotype 3 into genotype 1 increased the ability of the genotype 1 virus to grow in cell culture and we serially passaged the mutant and adapted it to grow more efficiently in cell culture. Ribavirin has been used to treat a small number of patients chronically-infected with genotype 3 HEV. In FY2013, we continued testing genotypes 1, 2, and 3 viruses for sensitivity to ribavirin in cell-culture. In collaboration with Dr. Neyts in Belgium, we created a luciferase-expressing genotype 3 virus and used it to study hepatitis E virus sensitivity to ribavirin and to interferon. A manuscript has been submitted to Hepatology.